Method for the recovery and purification of poxviruses from infected cells

ABSTRACT

The present invention relates to a method for the recovery of poxviruses, in particular modified Vaccinia virus Ankara (MVA), from infected cells. According to the present invention the virus-infected cells are subjected to a high-pressure homogenization to obtain a virus containing homogenate. The virus containing homogenate can be subjected to at least one purification step to obtain apoxvirus-enriched fraction. The invention further relates to the virus containing fraction and the virus containing homogenate obtained by the method according to the present invention.

[0001] The present invention relates to a method for the recovery ofpoxviruses, in particular modified Vaccinia virus Ankara (MVA), frominfected cells. According to the present invention the poxvirus-infectedcells are subjected to a high-pressure homogenization to obtain apoxvirus-containing homogenate. The poxvirus-containing homogenate canbe subjected to at least one purification step to obtain apoxvirus-enriched fraction. The invention further relates to thepoxvirus-containing fraction and the poxvirus-containing homogenateobtained by the method according to the present invention.

BACKGROUND OF THE INVENTION

[0002] The poxyiridae comprise a large family of complex DNA virusesthat replicate in the cytoplasm of vertebrate and invertebrate cells.The family of poxyiridae can be divided into the subfamilychordopoxyirinae (vertebrate poxviruses) and entomopoxyirinae (insectpoxviruses).

[0003] The chordopoxyirinae comprise several animal poxviruses(classified in different genera) of significant economical importance,such as camelpox viruses, sheeppox virus, goatpox virus oravipoxviruses, in particular fowlpoxvirus. For the vaccination oflivestock against sheeppox and goatpox attenuated live-virus andinactivated vaccines are available. For the vaccination of poultryrecombinant vaccines have been developed using fowlpox virus as avector.

[0004] Since fowlpoxvirus infects human cells it is assumed that it canalso be used as a vector to express heterologous genes in humans and toinduce a corresponding immune response. Fowlpoxviruses containing HIVgenes in the genome are disclosed in U.S. Pat. No. 5,736,368 and U.S.Pat. No. 6,051,410.

[0005] In humans the variola virus, a member of the genus Orthopoxvirus,was by far the most important poxvirus. Vaccinia virus, also a member ofthe genus Orthopoxvirus in the family of Poxyiridae, was used as livevaccine to immunize against smallpox. Successful worldwide vaccinationwith Vaccinia virus culminated in the eradication of variola virus (Theglobal eradication of smallpox. Final report of the global commissionfor the certification of smallpox eradication; History of Public Health,No. 4, Geneva: World Health Organization, 1980). Since that WHOdeclaration, vaccination has been discontinued for many years except forpeople at high risk of poxvirus infections (e.g. laboratory workers).Vaccination programs are again becoming of interest in view of the riskthat variola virus is used in biological warfare or by bioterrorists.

[0006] More recently, Vaccinia viruses have also been used to engineerviral vectors for recombinant gene expression and for the potential useas recombinant live vaccines (Mackett, M., Smith, G. L. and Moss, B.[1982] P.N.A.S. USA 79, 7415-7419; Smith, G. L., Mackett, M. and Moss,B. [1984] Biotechnology and Genetic Engineering Reviews 2, 383-407).This entails DNA sequences (genes), which code for foreign antigensbeing introduced, with the aid of DNA recombination techniques, into thegenome of the Vaccinia viruses. If the gene is integrated at a site inthe viral DNA which is nonessential for the life cycle of the virus, itis possible for the newly produced recombinant Vaccinia virus to beinfectious, that is to say able to infect foreign cells and thus toexpress the integrated DNA sequence (EP Patent Applications No. 83, 286and No. 110, 385). The recombinant Vaccinia viruses prepared in this waycan be used, on the one hand, as live vaccines for the prophylaxis ofinfectious diseases, on the other hand, for the preparation ofheterologous proteins in eukaryotic cells.

[0007] The use of Vaccinia virus as vector for the development ofrecombinant live vaccines has been affected by safety concerns andregulations. Most of the recombinant Vaccinia viruses described in theliterature are based on the Western Reserve strain of Vaccinia virus. Itis known that this strain has a high neurovirulence and is thus poorlysuited for use in humans and animals (Morita et al., Vaccine 5, 65-70[1987]). On the other hand the Modified Vaccinia virus Ankara (MVA) isknown to be exceptionally safe. MVA has been generated by longtermserial passages of the Ankara strain of Vaccinia virus (CVA) on chickenembryo fibroblasts (for review see Mayr, A., Hochstein-Mintzel, V. andStickl, H. [1975] Infection 3, 6-14; Swiss Patent No. 568, 392).Examples for MVA virus strains deposited in compliance with therequirements of the Budapest Treaty are strains MVA 572, MVA 575 andMVA-BN deposited at the European Collection of Animal Cell Cultures(ECACC), Salisbury (UK) with the deposition numbers ECACC V94012707,ECACC V00120707 and ECACC V00083008, respectively. MVA is distinguishedby its great attenuation that is to say by diminished virulence orinfectiosity while maintaining good immunogenicity. The MVA virus hasbeen analyzed to determine alterations in the genome relative to thewild type CVA strain. Six major deletions of genomic DNA (deletion I,II, III, IV, V, and VI) totaling 31,000 base pairs have been identified(Meyer, H., Sutter, G. and Mayr A. [1991] J. Gen. Virol. 72, 1031-1038).The resulting MVA virus became severely host cell restricted to aviancells.

[0008] Furthermore, MVA is characterized by its extreme attenuation.When tested in a variety of animal models, MVA was proven to beavirulent even in immunosuppressed animals. More importantly, theexcellent properties of the MVA strain have been demonstrated inextensive clinical trials (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B167, 375-390 [1987], Stickl et al., Dtsch. med. Wschr. 99, 2386-2392[1974]). During these studies in over 120,000 humans, includinghigh-risk patients, no side effects were associated with the use of MVAvaccine. Recombinant MVA useful as vaccines have already beenconstructed and used in clinical trials. WO 98/13500 discloses arecombinant MVA containing and capable of expressing one or more DNAsequences encoding dengue virus antigens. The foreign DNA sequences wererecombined into the viral DNA at the site of a naturally occurringdeletion in the MVA genome.

[0009] Before using poxviruses or recombinant poxvirus for vaccinationit is necessary to purify the virus to a certain extent in order to meetregulatory requirements. The traditional way to purify poxviruses, inparticular MVA and recombinant MVA is as follows: in a first step cellssusceptible to infection with the respective poxvirus are cultivated. Incase of MVA the susceptible cells are i.a. chicken embryo fibroblasts.The susceptible cells are infected with the poxvirus and cultivated fora time period sufficient to allow the generation of virus progeny. Thecells then are frozen and thawed in order to detach the cells from theculture vial surface and to partially disrupt the cells. The mixture ofintact and disrupted cells is spun down. Ultrasound is used to produce ahomogenate. Virus is purified from the homogenate by sucrose cushioncentrifugation (Joklik WK. “The purification of four strains ofpoxvirus” Virology 1962; 18:9-18). The key step in this process is thehomogenization by using ultrasound (Hedström, K. G. and Lindberg, U., Z.Immun. Forsch. 1969 137:421-430; Stickl, H., Korb, W. andHochstein-Mintzel, V., Zbl. Bakt., I. Abt. Orig. (1970), 215, 38-50). Inindustrial processes it is preferred that all process steps are easy tocontrol and reproducible. However the disadvantage in using ultrasoundto homogenize the viruscell suspension is that the ultrasound step isdifficult to reproduce in an identical manner, difficult to adjust andit is difficult to scale up the process from laboratory to industrialscale.

OBJECT OF THE INVENTION

[0010] Thus, it is an object of the invention to provide a method forthe recovery of poxviruses, in particular of Vaccinia viruses, such asstrain MVA, from poxvirus infected cells, wherein the homogenization ofthe infected cells is reproducible, easy to control and allows an easyscaling up from laboratory to industrial scale.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention concerns a method for the recovery ofpoxviruses, in particular vaccinia viruses, such as strain Elstree orModified Vaccinia Virus Ankara (MVA), from infected cells. The methodaccording to the present invention for the recovery of poxvirus frominfected cells comprises the step of subjecting the infected cells to ahigh-pressure homogenization to obtain a poxvirus-containing homogenate.

[0012] It was unexpected that intact and infectious poxviruses can berecovered by the method according to the present invention.High-pressure homogenization is commonly used for the destruction ofcellular and subcellular structures in order to isolate proteins orlipids from eukaryotic and prokaryotic cells. U.S. Pat. No. 3,983,008discloses a method of extracting useful components from microbial cellsby using high pressure homogenizations. The extracted compounds wereyeast proteins, bacterial enzymes and yeast lipids. DE 19918619discloses the use of high-pressure homogenization to isolate HbsAg fromyeast cells. U.S. Pat. No. 4,255,521 describes a process for extractingglucose isomerase from microorganism cells by high pressure release.High-pressure homogenization has also been used for the isolation ofvirus-like particles (VLP) from recombinant Saccharomyces cerevisiae(Milburn and Dunnill (1994) Biotechnology and Bioengineering 44,736-744) and for the production and purification of adenoviral vectors(U.S. Pat. No. 6,194,191). VLPs and Adenoviruses are non-enveloped andrather small and simple viruses that, thus, resemble cellular proteinstructures. Therefore, it is not astonishing that the high-pressurehomogenization that has been shown to be suitable for the isolation ofproteins from cells can also be used for the isolation of Adenovirusesand VLPs from eukaryotic cells. In contrast, the intracellular maturepoxvirus virions (IMV, see below, see Fields et al., Fields Virology,1996, Lippincott-Raven publishers, Philadelphia, USA, ISBN0-7817-0253-4, chapter 83, pages 2654-5) have a very complex morphologythat involves inter alia lipid membranes. The morphology and physicalproperties of a poxvirus IMV are in some aspects more closely related tothe morphology and the physical properties of a cell than to those of anon-enveloped virus. Consequently, it was expected that the conditionsused for the disruption of cells by using high-pressure homogenizationwould also lead to the disruption of poxviruses. Thus, it was asurprising result that high-pressure homogenization disrupts cells butleaves intact a sufficient amount of poxviruses that may be furtherpurified. In other words, one would not have assumed that high-pressurehomogenization could be used in a method for the recovery of poxvirusesfrom infected cells. Indeed, the known method for the recovery ofpoxviruses from infected cells uses ultrasound for homogenization, whichis a rather gentle way of homogenization.

[0013] In contrast to the recovery methods that use ultrasound themethod according to the present invention allows a reproduciblehomogenization of infected cells; the method is easy to control and itis easy to scale up the process from a laboratory to an industrialscale.

[0014] In the context of the present invention the term “poxvirus”refers to any virus belonging to the family poxyiridae. The methodaccording to the present invention is preferably carried out withpoxyiridae of the subfamily chordopoxyirinae, more preferably of thegenera orthopoxvirus, avipoxvirus, capripoxvirus and suipoxvirus. Mostpreferably the invention concerns a method for the recovery andpurification of poxviruses selected from the group consisting ofVaccinia virus, goatpoxvirus, sheeppoxvirus, canarypoxvirus andfowlpoxvirus. Particularly preferred is Vaccinia virus. Examples forvaccinia virus strains used in the method according to the presentinvention are the strains Elstree, Wyeth, Copenhagen, Temple of Heaven,NYCBH, Western Reserve. The invention is not restricted to thosespecifically mentioned vaccinia virus strains but may instead be usedwith any vaccinia virus strain. A preferred example for a Vaccinia virusstrain is the modified Vaccinia virus strain Ankara (MVA). A typical MVAstrain is MVA 575 that has been deposited at the European Collection ofAnimal Cell Cultures under the deposition number ECACC V00120707. Mostpreferred is MVA-BN or a derivative thereof. MVA-BN has been describedin WO 02/42480 (PCT/EP01/13628). Said international applicationdiscloses biological assays allowing to evaluate whether a MVA strain isMVA-BN or a derivative thereof and methods allowing to obtain MVA-BN ora derivative thereof. The content of this application is included in thepresent application by reference. MVA-BN has been deposited at theEuropean Collection of Animal Cell Cultures with the deposition numberECACC V00083008.

[0015] The viruses to be recovered may be native viruses, attenuatedviruses or recombinant viruses.

[0016] The term “recombinant virus” refers to any virus having insertedinto the viral genome a heterologous gene that is not naturally part ofthe viral genome. A heterologous gene can be a therapeutic gene, a genecoding for an antigen or a peptide comprising at least one epitope toinduce an immune response, an antisense expression cassette or aribozyme gene. Methods to obtain recombinant viruses are known to aperson skilled in the art. The heterologous gene is preferably insertedinto a nonessential region of the virus genome. In another preferredembodiment of the invention, the heterologous nucleic acid sequence isinserted at a naturally occurring deletion site of the MVA genome(disclosed in PCT/EP96/02926).

[0017] An “attenuated virus” is a virus that upon infection of the hostorganism results in a lower mortality and/or morbidity compared to thenon attenuated parent virus. An example for an attenuated Vaccinia virusis strain MVA, in particular MVA575 and MVA-BN.

[0018] Poxviruses, such as Vaccinia virus, are known to exist in twodifferent forms: poxvirus attached to cellular membranes in thecytoplasm of the infected cells (intracellular mature virions (IMV)) andviruses that have been externalized (extracellular enveloped virions(EEV)) (Vanderplasschen A, Hollinshead M, Smith GL “Intracellular andextracellular vaccinia virions enter cells by different mechanisms” J.

[0019] Gen. Virol. (1998), 79, 877887). IMVs and EEVs are bothinfectious but morphologically different since EEV contain an additionallipoprotein envelope. Under normal circumstances IMV particles are moreabundant than EEV, but in the method according to the invention bothtypes of particles can be obtained.

[0020] The starting materials for the homogenization step according tothe present invention are cells infected with the respective poxvirus.The term “infected cells” used to define the starting material for thehomogenization according to the present invention refers to intact cellsinfected with the respective virus, to parts and fragments of infectedcells to which the respective poxvirus is attached or to a mixture ofintact cells and lysed/disrupted cells. Examples for a part or afragment of infected cells are cell membranes of disrupted/lysed cellsto which the respective poxvirus is attached. The starting material mayalso contain free virus particles that are neither attached to cellularmembrane nor located intracellularly.

[0021] In order to obtain the infected cells that are the startingmaterial for the method according to the present invention eukaryoticcells are infected with the respective poxvirus. The eukaryotic cellsare cells that are susceptible to infection with the respective poxvirusand allow replication and production of infectious virus. Such cells areknown to the person skilled in the art for all poxviruses. For MVA andvaccinia virus strain Elstree an example for this type of cells arechicken embryo fibroblasts (CEF) (Drexler I., Heller K., Wahren B.,Erfle V. and Sutter G.

[0022] “Highly attenuated modified vaccinia Ankara replicates in babyhamster kidney cells, a potential host for virus propagation, but not invarious human transformed and primary cells” J. Gen. Virol. (1998), 79,347-352). CEF cells can be cultivated under conditions known to theperson skilled in the art. Preferably the CEF cells are cultivated inserum-free medium in stationary flasks or roller bottles. The incubationpreferably takes place 48 to 96 hours at 37° C. ±2° C. For the infectionpoxviruses are preferably used at a multiplicity of infection (MOI) of0,05 to 1 TCID₅₀ and the incubation preferably takes place 48 to 72hours at 37° C.±2° C.

[0023] Progress of infection can be observed by looking at cytopathiceffects (CPE), typically appearing by significant rounding of theinfected cells.

[0024] The present invention allows the recovery of poxviruses, such asElstree or MVA from infected cells. By the term “recovery” it is meantthat the method of the present invention allows to disrupt poxvirusinfected cells and/or to detach the poxviruses from the cellularmembranes to which they are usually bound, to such an extend that afurther purification of the poxvirus becomes feasible. Thus, the productof the recovery of poxviruses from infected cells (referred to as“poxvirus-containing homogenate” in the present application) is ahomogenous mixture of free poxvirus and cellular detritus containingonly minor amounts of intact, undisrupted cells and virus bound tocellular membranes.

[0025] If the infected cells are cells that can be cultivated insuspension culture the infected cells can easily be harvested bycentrifugation.

[0026] If the infected cells are more or less intact adherent cells theyshould be harvested, i.e. removed from the culture vial, beforesubjecting them to the high-pressure homogenization. Such methods areknown to the person skilled in the art. Useful techniques are mechanicmethods (e.g. by using a rubber cell scraper), physical methods (e.g.freezing below −15° C. and thawing the culture vessels above +15° C.) orbiochemical methods (treatment with enzymes, e.g. trypsin, to detach thecells from the culture vessel). If enzymes are used for this purpose theincubation time should be controlled, since these enzymes may alsodamage the virus during incubation.

[0027] In the method according to the present invention the infectedcells, more specifically the harvested infected cells are then subjectedto a high pressure homogenization step. In the present specification theterm “high pressure homogenization” is sometimes abbreviated as “HPH”.The high-pressure homogenization has a dual effect. On the one hand thehigh-pressure homogenization leads to the disruption of intact cells.Thus, the IMVs are freed and become available for a furtherpurification. On the other hand the high-pressure homogenization has theeffect that the poxviruses are detached from cell membranes or at leastthat the size of the cell-membrane-virus aggregates is reduced.

[0028] Again, this simplifies the further purification of the poxvirus.

[0029] The person skilled in the art is familiar with the generalprinciple of high-pressure homogenization (White MD, Marcus D.,“Disintegration of microorganisms”, Adv. Biotechnol. Processes 1988;8:51-96). HPH Systems are based on the use of high pressure to force asample through a small fixed orifice at high speed under controlled andrepeatable conditions. In the present description the terms “jet”,“orifice” and “nozzle” are used interchangeably.

[0030] The heart of each cell disrupter is a disruption head. Thedisruption head preferably consists of (I) a high pressurechamber/cylinder, (II) a high pressure piston which moves into thechamber/cylinder and thereby increases the pressure in thechamber/cylinder and (Ill) a nozzle/jet through which the chambercontent is ejected. The ejected chamber content is directed to a targetsurface such as a piece of metal that preferably has a heat exchangesurface allowing cooling. To collect the disrupted chamber content thesystem is provided with means for collection of the disrupted sample(termed “collection chamber”). A typical high pressure homogenizationunit is Basic Z+from Constant Cell Disruption Systems (Low March,Daventry, Northants, NN114SD, United Kingdom).

[0031] In a preferred embodiment there are three stages to effect celldisruption using the high-pressure homogenization system. (I) A sampleis introduced into the high pressure cylinder/chamber. Then the pressurein the cylinder/chamber is built up. To this end the high pressurepiston descends. (II) The piston then forces the sample through thenozzle at high speed. The rapid transfer of the sample from a region ofhigh pressure to one of low pressure causes cell disruption. (III) Thesample hits the target and is spread radially across the cooled heatexchange surface. The product then flows into a chamber for collection.The hydraulics is recharged and the cycle continues.

[0032] At the end of the process the ejected homogenate is collected inan appropriate vial, depending on the volume.

[0033] For the recovery of poxviruses according to the present inventionthe nozzle should have a diameter in the range of 0.10 to 0.6 mm, 0.15to 0.6 mm, 0.15 to 0.50 mm, preferably in the range of 0.15 to 0.40 mm,0.20 to 0.50 mm, more preferably 0.20 to 0.40 mm, most preferably 0.25mm to 0.35 mm. Examples for most preferred diameters are 0.25 mm and0.35 mm.

[0034] The pressure in the pressure chamber/cylinder is set to a valuein the range of 200 to 1000 bar, preferably 400 to 1000 bar, 200 to 800bar, more preferably 400 to 800 bar, 600 to 1000 bar, even morepreferably 600 to 900 bar, most preferably 700 to 900 bar. The mostpreferred pressure is 800 bar.

[0035] The temperature of the homogenate at the outlet preferably shouldnot exceed +25° C., and should preferably be below 15° C., mostpreferably below 10° C.

[0036] The method according to the present invention can be scaled-upalmost linear and can be run either as batch or as continuous process.

[0037] In a batch process each batch of the cells to be homogenized canbe subjected to the high pressure homogenization step once or severaltimes. Preferably each batch is subjected to the homogenization step oneto three times, most preferably only once.

[0038] The success of the homogenization step may preferably be checkedby determination of the virus titer (equivalent to the number ofinfectious virus particles, measured either in tissue culture infectiousdose (TCID₅₀), or plaque forming units (pfu)) of the starting materialas defined above and of the material obtained after the homogenizationstep. In other words the virus titer is determined before and after thehigh-pressure homogenization. The starting material comprises more orless intact cells and a rather high percentage of large aggregatescomprising poxvirus particles bound to cellular membranes. If such amaterial is used for the determination of the viral titer the obtainedtiter is lower than the actual number of infectious particles. This isdue to the fact that the test systems used for the determination of theviral titer are usually cell culture systems (such as the system asdisclosed in Example 2) in which the number of infected cells or thenumber of plaques is counted. Such a system can not distinguish betweena positive result that is due to the infection of a cell by just onevirus particle and the infection of cells e.g. by a large aggregate ofviruses bound to cellular membranes. After the high-pressurehomogenization the poxviruses become detached from the cellularmembranes and/or the size of cell membrane-virus aggregates issignificantly reduced, which leads to a larger number of smalleraggregates. If this material is used for the determination of the titerthe obtained results are higher, even if the actual amount of infectiousvirus particles has not changed.

[0039] Thus, the success of the homogenization is preferably reflectedby at least an equal or higher TCID₅₀/ml (“TCID” is the abbreviation of“tissue culture infectious dose”) of the homogenate compared to thestarting material. In the example section it is shown in detail how theTCID₅₀/ml value can be determined. Alternatively, the quality and thesuccess of the high-pressure homogenization can be determined byelectron microscopy.

[0040] For further purification the obtained homogenate is subjected toat least one purification step to obtain a poxvirus-enriched fraction.In particular, these steps can be carried out for a vaccinia viruscontaining homogenate if it is intended to further purify said vacciniavirus.

[0041] The purification step inter alia can be

[0042] batch centrifugation (e.g. using sucrose cushions) orcontinuous-flow ultracentrifugation (sucrose gradients) (Hilfenhaus, J.,Köhler, R. and Gruschkau, H., J. Biol. Stand. (1976) 4, 285-293;Madalinski, W., Bankovski, A. and Korbecki, M., Acta Virol. (1977) 21,104-108),

[0043] ultrafiltration (e.g. crossflow filtration using a membrane witha pore size bigger than 500 kDa, but equal or smaller than 0,1 μm),

[0044] column chromatography (e.g. ion exchange, hydrophobicinteraction, size exclusion or a combination) (Hedström, K. G. andLindberg, U., Z. Immun. Forsch. 1969 137:421-430; Stickl, H., Korb, W.and Hochstein-Mintzel, V., Zbl. Bakt., l. Abt. Orig. (1970), 215, 38-50)or

[0045] a combination of all of the above (Masuda, N., Ellen, R. P. andGrove, D. A., J. Bacteriol. (1981), 147(3), 1095-1104).

[0046] Any other purification methods known to the person skilled in theart are also within the scope of the present invention.

[0047] Preferably the purification step is an ultrafiltration step. Mostpreferably the ultrafiltration is a cross-flow filtration. The principleof cross-flow filtration is known to the person skilled in the art. See,e.g., Richards, G. P. and Goldmintz, D., J. Virol. Methods (1982), 4(3),pages 147-153. “Evaluation of a cross-flow filtration technique forextraction of polioviruses from inoculated oyster tissue homogenates”.

[0048] Although one purification step might be sufficient to meetregulatory requirements for biopharmaceutical products, two or more ofthe above mentioned purification steps can be combined in order toobtain an even more pure product.

[0049] In the most preferred embodiment the first purification step is across-flow-filtration followed by at least one column chromatographystep. Most preferably the column chromatography step is ion exchange orhydrophobic interaction.

[0050] The obtained virus enriched fraction is optionally freeze-dried.Methods of freeze-drying are known to the person skilled in the art (DayJ. and McLellan M., Methods in Molecular Biology (1995), 38, HumanaPress, “Cryopreservation and freeze-drying protocols”).

[0051] The invention further concerns the poxvirus-enriched fractionand/or the poxvirus-containing homogenate obtained by the method forrecovery of poxviruses according to the present invention, i.e. themethod that comprises the step of subjecting the infected cells tohigh-pressure homogenization. In particular the invention concerns thepoxvirus-enriched fraction obtained by the recovery/purification methodaccording to the present invention, i.e. the method in which thepoxvirus-containing homogenate obtained by HPH is subjected to at leastone purification step. The poxvirus may be any poxvirus as definedabove. In particular the poxvirus according to the present invention isa vaccinia virus as, such any strains that are suitable for vaccination,in particular strain Elstree or modified vaccinia virus Ankara, mostpreferably MVA-BN.

[0052] The poxvirus containing homogenate and/or the poxvirus-enrichedfraction obtained by a process according to the present invention thatcomprises a HPH step is characterized by a very high free-IMV poxvirusto EEV poxvirus ratio. The term “free IMV” is used for IMVs that havebeen detached from the cellular membranes after, before or duringdisruption of the infected cells and that therefore can be furtherpurified. In all industrial processes for the preparation of poxvirusesthe starting material comprises the infected cells as well as theculture supernatant. Thus, the starting material comprises IMVpoxviruses contained in the infected cells as well as EEV poxviruseswhich are mainly found in the supernatant. The known methods for thedisruption of cells and the subsequent homogenization (e.g. by usingultrasound) do not as efficiently disrupt the cells and/or detach theIMV poxviruses from cellular debris as high pressure homogenization. Inother words most of the IMV remain bound to cellular membranes anddebris. Thus, the ratio of free IMV to EEV is lower than in the methodaccording to the present invention. In the method using ultrasound thisratio does not change significantly during the further purificationsteps since the cellular debris to which IMVs are still bound is usuallyremoved. In contrast to the known recovery methods for poxviruses therecovery method according to the present invention results in a veryeffective disruption of the infected cells and the IMVs are veryeffectively detached from the cell membranes. Thus, the overall amountof free IMVs that become available for further purification is higherthan for the methods known in the prior art and consequently also theratio of free IMV to EEV poxviruses is higher.

[0053] The poxvirus-containing homogenate and/or the poxvirus-enrichedfraction obtained by the method according to the present invention areuseful as vaccines.

[0054] If the poxvirus-containing homogenate and/or thepoxvirus-enriched fraction comprise unmodified poxviruses or attenuatedpoxviruses, such as vaccinia virus strains Elstree or MVA, it can beused for vaccination against poxvirus infections. E.g. a viruscontaining homogenate and/or the virus-enriched fraction that comprisesvaccinia viruses such as strain Elstree or MVA, in particular MVA-BN canbe used as a vaccine against smallpox infections.

[0055] If the poxvirus-containing homogenate and/or thepoxvirus-enriched fraction comprises a poxvirus that contains andexpresses one or more heterologous gene(s) the poxvirus-containinghomogenate and/or the poxvirus-enriched fraction can further be used tovaccinate animals including human beings against the protein expressedby the heterologous gene(s).

[0056] For the preparation of a vaccine, the poxvirus-containinghomogenate and/or the poxvirus-enriched fraction obtained by the methodaccording to the present invention are converted into a physiologicallyacceptable form. This can be done based on the experience in thepreparation of poxvirus vaccines used for vaccination against smallpox(as described by Stickl, H. et al. [1974] Dtsch. med. Wschr. 99,2386.2392). For example, the poxvirus-containing homogenate and/or thepoxvirus-enriched fraction are stored at −80° C. with a titer of 5×10⁸TCID₅₀/ml formulated in about 10 mM Tris, 140 mM NaCl pH 7.4. For thepreparation of vaccine shots, e.g., 10³·10⁹ TCID₅₀ of the virus arelyophilized in phosphate-buffered saline (PBS) in the presence of 2%peptone and 1% human albumin in an ampoule, preferably a glass ampoule.Alternatively, the vaccine shots can be produced by stepwisefreeze-drying of the virus in a formulation. This formulation cancontain additional additives such as mannitol, dextran, sugar, glycine,lactose or polyvinylpyrrolidone or other additives such as antioxidantsor inert gas, stabilizers or recombinant proteins (e.g. human serumalbumin) suitable for in vivo administration. A typical virus containingformulation suitable for freeze-drying comprises 10 mM Tris-buffer, 140mM NaCl, 18.9 g/l Dextran (MW 36000-40000), 45 g/l Sucrose, 0.108 g/lL-glutamic acid mono potassium salt monohydrate pH 7.4. The glassampoule is then sealed and can be stored between 4° C. and roomtemperature for several months. However, as long as no need exists theampoule is stored preferably at temperatures below −20° C.

[0057] For vaccination the lyophilized or freeze-dried product can bedissolved in 0.1 to 0.5 ml of an aqueous solution, preferablyphysiological saline or Tris buffer, and administered eithersystemically or locally, i.e. parenterally, intramuscularly or by anyother path of administration know to the skilled practitioner. The modeof administration, the dose and the number of administrations can beoptimized by those skilled in the art in a known manner. Most preferredfor poxvirus vectors is subcutaneous or intramuscular administration.

[0058] The invention further relates to a method for the vaccination ofanimals including humans comprising inoculating an animal, including ahuman, in need thereof with a poxvirus-containing homogenate or apoxvirus-enriched fraction obtained by the method according to thepresent invention.

SUMMARY OF THE INVENTION

[0059] The invention inter alia comprises the following, alone or incombination:

[0060] Method for the recovery of poxvirus from infected cellscomprising the step of subjecting the infected cells to a high-pressurehomogenization to obtain a poxvirus-containing homogenate.

[0061] Method as above characterized in that the poxvirus is selectedfrom the group consisting of orthopoxviruses, avipoxviruses,suipoxviruses and capripoxviruses

[0062] Method as above characterized in that the poxvirus is selectedfrom the group consisting of vaccinia virus, goatpoxvirus,sheeppoxvirus, canarypoxvirus and fowlpoxvirus

[0063] Method as above characterized in that the vaccinia virus ismodified vaccinia virus strain Ankara (MVA), in particular MVA-BN withthe deposition number ECACC V00083008.

[0064] Method as above characterized in that the poxvirus is arecombinant pox virus.

[0065] Method as above characterized in that the high-pressurehomogenization is carried out by putting the infected cells into a highpressure chamber, increasing the pressure in the chamber and ejectingthe infected cells through a nozzle.

[0066] Method as above characterized in that the pressure in the chamberis increased to a value in the range of 200 to 1000 bar.

[0067] Method as above characterized in that the nozzle has a diameterin the range of 0.10 to 0.6 mm.

[0068] Method as above characterized in that the poxvirus-containinghomogenate is subjected to at least one purification step to obtain apoxvirus-enriched fraction.

[0069] Method as above characterized in that the at least onepurification step is an ultrafiltration step.

[0070] Method as above characterized in that the ultrafiltration is across-flow-filtration.

[0071] Method as above characterized in that in the crossflow-filtrationstep a membrane is used that has a pore size bigger than 500 kDa butequal or smaller than 0.1 μm.

[0072] Method as above characterized in that subsequent to theultrafiltration at least one column chromatography step is carried outMethod as above characterized in that the obtained poxvirus-containinghomogenate or poxvirus-enriched fraction is freeze-driedPoxvirus-containing homogenate or poxvirus-enriched fraction obtained bya method as defined above.

[0073] Poxvirus-containing homogenate or poxvirus-enriched fraction asabove as vaccine.

[0074] Use of the poxvirus-containing homogenate or poxvirus-enrichedfraction as above for the preparation of a vaccine.

[0075] Method for the vaccination of an animal, including a human, inneed thereof characterized by the administration of apoxvirus-containing homogenate or poxvirus-enriched fraction or avaccine as defined above to the animal body.

Figure Legends

[0076]FIG. 1: CEF cells infected with MVA-BN were subjected to afreezing-thawing cycle to obtain a virus-cell suspension. The virustiter of the suspension was determined as described in example 2 withoutfurther purification of the cell-virus-suspension (“O-value”). Thevirus-cell-suspension was subjected to a homogenization method knownfrom the prior art by using ultrasound (“Sonifier”) or to thehomogenization method according to the present invention (“HPH”) asdescribed in Example 1. In the HPH step the pressure was 800 bar and thenozzle had a diameter of 0.25 mm. At the end of the homogenization stepthe virus titer was again determined.

EXAMPLE(S)

[0077] The following example(s) will further illustrate the presentinvention. It will be well understood by a person skilled in the artthat the provided example(s) in no way may be interpreted in a way thatlimits the applicability of the technology provided by the presentinvention to this example(s).

Example 1 Homogenization of Poxvirus-Cell-Suspensions by Using a HighPressure Homogenizer

[0078] Chicken embryo fibroblasts (CEF) cultivated in roller flasks havebeen infected with MVA-BN (ECACC V00083008). The infected cells weresubjected to freezing and thawing to obtain a virus-cell-suspension.

[0079] A Basic Z+ homogenizer from Constant Cell Disruption Systems (LowMarch, Daventry, Northants, NN114SD, United Kingdom) was loaded with 50ml of the crude virus-cell-suspension for each run. In order to identifythe optimal homogenization conditions nozzle diameters in the range of0.18 to 0.40 mm and pressures in the range of 200 to 1000 bar weretested. The crude suspension was subjected to the high pressurehomogenization one, two or three times. The homogenizer was usedaccording to the instructions of the manufacturer. The evaluation of themethod was executed by monitoring the titer as described in example 2.

[0080] In preliminary studies it was found that jet diameters largerthan 0.4 mm have no positive influence on the homogenization results.With the nozzle diameters of 0.18 mm, 0.25 mm and 0.35 mm the bestresults were obtained by subjecting the viruscell suspension once to apressure of 800 bar. At these conditions no major difference in terms oftiter have been observed.

[0081] The method according to the present invention was compared to thedirect flow-through ultrasound treatment known from the prior art. Theresults are summarized in FIG. 1. Compared to the ultrasound treatmentthe method according to the present invention resulted in a higher virustiter and a better homogeneity of the suspension, so that it is moresuitable for further downstream processing.

Example 2 Titration of Modified Vaccinia Virus Ankara (MVA)

[0082] The titration of Modified Vaccinia virus Ankara (MVA) isperformed in a TCID₅₀-based assay using 10-fold dilutions in a 96-wellformat. At the endpoint of the assay, infected cells are visualisedusing an anti-vaccinia virus antibody and an appropriate stainingsolution.

[0083] 2-3 day old primary CEF (chicken embryo fibroblasts) cells arediluted to 1×10⁵ cells/ml in 7% RPMI. 10 fold dilutions are done with 8replicates per dilution. Following dilution, 100 μl are seeded per wellof 96-well plates. Cells are then incubated over night at 37° C. and 5%CO₂.

[0084] Dilutions of the virus containing solutions are made in 10-foldsteps (10⁻to 10⁻¹² as appropriate) using RPMI without fetal calf serum.Then, 100 μl of every virus sample is added to the cell containingwells.

[0085] The 96-well-plates are incubated at 37° C. with 5% CO₂ for 5 daysto allow infection and viral replication.

[0086] Cells are stained 5 days after infection with a vaccinia virusspecific antibody. For the detection of the specific antibody, ahorseradish peroxidase (HRP) coupled secondary antibody is used. The MVAspecific antibody is an anti-vaccinia virus antibody, rabbit polyclonal,IgG fraction (Quartett, Berlin, Germany #9503.2057). The secondaryantibody is anti-rabbit IgG antibody, HRP coupled goat polyclonal(Promega, Mannheim, Germany, # W4011). The colour reaction is carriedout according to known techniques.

[0087] Every well with cells that are positive in the colour reaction ismarked as positive for the calculation of the TCID₅₀.

[0088] The titre is calculated by using the formula of Spearman [1] andKaerber [2]. Because all assay parameters are kept constant, thefollowing simplified formula is used:${{Virus}{\quad \quad}{{titre}\quad\left\lbrack {{TCID}_{50}/{ml}} \right\rbrack}} = 10^{\lbrack{{a + 1},{5 + \frac{x_{a}}{8} + \frac{x_{b}}{8} + \frac{x_{c}}{8}}}\rbrack}$

[0089] a=dilution factor of last column, in which all eight wells arepositive

[0090] x_(a)=number of positive wells in column a+1

[0091] x_(b)=number of positive wells in column a+2

[0092] x_(c)=number of positive wells in column a+3

1. Method for the recovery of poxvirus from infected cells comprisingthe step of subjecting the infected cells to a high-pressurehomogenization to obtain a poxvirus-containing homogenate.
 2. Methodaccording to claim 1 characterized in that the poxvirus is selected fromthe group consisting of orthopoxviruses, avipoxviruses, suipoxvirusesand capripoxviruses
 3. Method according to claim 1 characterized in thatthe poxvirus is selected from the group consisting of vaccinia virus,goatpoxvirus, sheeppoxvirus, canarypoxvirus and fowlpoxvirus
 4. Methodaccording to claim 3 characterized in that the vaccinia virus is Elstreeor modified vaccinia virus strain Ankara (MVA), in particular MVA-BNwith the deposition number ECACC V00083008.
 5. Method according to claim1 characterized in that the poxvirus is a recombinant poxvirus. 6.Method according to claim 1 characterized in that the high-pressurehomogenization is carried out by putting the infected cells into a highpressure chamber, increasing the pressure in the chamber and ejectingthe infected cells through a nozzle.
 7. Method according to claim 6characterized in that the pressure in the chamber is increased to avalue in the range of 200 to 1000 bar.
 8. Method according to claim 6characterized in that the nozzle has a diameter in the range of 0.10 to0.6 mm.
 9. Method according to claim 1 characterized in that thepoxvirus-enriched containing homogenate is subjected to at least onepurification step to obtain a poxvirus-enriched fraction.
 10. Methodaccording to claim 9 characterized in that one of the at least onepurification steps is an ultrafiltration step.
 11. Method according toclaim 10 characterized in that the ultrafiltration is across-flow-filtration.
 12. Method according to claim 11 characterized inthat in the cross-flow-filtration step a membrane is used that has apore size bigger than 500 kDa but equal or smaller than 0.1 gm. 13.Method according to claim 10 characterized in that subsequent to theultrafiltration at least one column chromatography step is carried out.14. Method according to claim 1 characterized in the poxvirus-containinghomogenate or the poxvirus-enriched fraction is freeze-dried. 15.Poxvirus-enriched fraction or poxvirus-containing homogenate obtained bythe method according to claim
 1. 16. Poxvirus-enriched fraction orpoxvirus-containing homogenate according to claim 15 as vaccine.
 17. Useof the poxvirus-enriched fraction or poxvirus-containing homogenateaccording to claim 15 for the preparation of a vaccine.
 18. Method forthe vaccination of an animal, including a human, in need thereofcharacterized by the administration of a vaccine according to claim 16to the animal body.